Monoclonal antibodies (mAbs) and related products constitute one of the most rapidly growing classes of human therapeutics. These large (˜150 kDa) proteins contain two identical (˜50 kDa) heavy chains and two identical (˜25 kDa) light chains. They also contain 16 or 17 disulfide bonds that maintain the three dimensional structure of the molecule. Different mAbs differ greatly in the sequence of their variable regions near the N-termini of both light and heavy chains. The most variable parts are complementarity-determining regions (CDRs), which are unique to individual mAbs and are responsible for the diversity and specificity of antibody bonding. Changes to the mAb structure introduced during the manufacturing process or storage can change the therapeutic efficacy, clearance, and immunogenicity properties of the protein and thus alter drug safety (11-14).
Therapeutic antibodies can have heterogeneities resulting from various modifications that occur during different stages of production, such as mutations, C-terminal lysine processing, pyroglutamic acid formation, oxidation, amidation, deamidation, glycosylation, and disulfide linkages. Identification of the primary sequence of therapeutic mAbs, as well as elucidation of the N-glycan structures, disulfide linkages and other PTMs, is critical for the evaluations of drug safety, efficacy, stability, as well as understanding the structure/function relationships. The demands for characterization of therapeutic mAbs are increasing with the rapid development of mAb-based pharmaceuticals. Moreover, the ability to readily generate such structure/function information with respect to a reference mAb would greatly accelerate the market entry of mAbs that could be deemed biosimilar to the reference mAb.
Widely adopted methods for protein structural characterizations by mass spectrometry (MS) involve a “bottom-up” approach. These methods are associated with complete tryptic digestion of the protein(s) into smaller peptides (<3000 Da) prior to MS analyses. Although useful for tandem MS (MS/MS, also referred to as MS2) analysis, small tryptic peptides often result in problems such as high sample complexity, difficulties in assigning peptides to specific gene products, and loss of combinatorial post-translational modifications (PTMs) information. Recent years have seen efforts toward achieving direct MS analysis of intact proteins (often called “top-down” MS). This approach aims to overcome the above issues by providing an overview of the entire protein sequence and PTMs. However, intact protein MS is still far from maturity in terms of being able to characterize large proteins. This is in part due to reasons such as inefficient gas-phase protein fragmentation and complex fragment ions that restrict efficient data interpretation. For example, the reported highest sequence coverage of intact therapeutic monoclonal antibodies (immunoglobulin G, 150 kDa) is no more than 35%, obtained by either ETD Orbitrap Fourier transform (FT) MS (Tsybin MCP 2012) or electron-capture dissociation (ECD) on a custom-built 9.4 T FT ion cyclotron resonance mass spectrometer (Marshall, Anal Chem. 2013, 85, 4239-4246).
There is a long felt need in the art for compositions, methods, and apparatuses useful for rapid sequence analysis of proteins, identification of post-translational modifications, and localization of disulfide bonds. The present invention satisfies these needs.